Arenaviruses are endemic in rodent populations and a number of species can be transmitted to humans to cause severe life-threatening hemorrhagic fevers. Public health and US security interests have driven the development of a classically derived live-attenuated virus vaccine to protect against Junn virus, the causative agent of Argentine hemorrhagic fever. Despite its use in Argentina, the Candid#1 vaccine has not been licensed in the US, in part due to concerns regarding the stability of attenuation, which relies on a single F427I mutation in the virus envelope glycoprotein (GPC). The current proposal seeks to capitalize on our understanding of GPC structure and function to design and characterize recombinant Candid#1 (rCan) viruses that enhance vaccine safety without compromising immunogenicity and protective efficacy. We hypothesize that epistatic interactions between GPC subunits can be leveraged to provide an evolutionary barrier against reversion of the attenuating mutation, and that attenuation can be further strengthened by incorporating well- characterized and genetically stable GPC deletions to minimize the consequences of reversion to F427. By characterizing rCan viruses that embody these strategies, we aim to enhance safety in a second-generation rCan vaccine. Towards this goal, we will pursue the following specific aims: Aim 1. Determine whether epistatic interactions between the SSP and GP2 subunits in GPC can be used to genetically fix the attenuating F427I substitution in rCan. The stable signal peptide (SSP) and GP2 fusion subunits in the tripartite GPC complex interact to sense endosomal pH and trigger membrane fusion. We have shown that GPC bearing a K33S mutation in SSP is unable to mediate membrane fusion or support viable virus, unless the mutation is paired with the attenuating F427I substitution in GP2. Indeed, K33S rCan is genetically stable in cell culture under conditions that drive I427F reversion in wild-type rCan. In this Aim, we will produce and validate K33S rCan for in vivo studies of vaccine attenuation and efficacy. Aim 2. Design and characterize rCan variants bearing redundant and genetically stable attenuation mutations. We have demonstrated that the ?G2 deletion in GPC eliminates SSP myristoylation and engenders coordinate reductions in GPC fusion activity and rCan specific infectivity. We have also identified a 7-amino-acid deletion in the fusion peptide of GP2 that supports viable ?FP rCan. We will characterize rCan viruses bearing these deletions in cell culture towards designing additional layers of attenuation. Aim 3. Assess genetic stability, attenuation and protective immunity of engineered rCan variants using complementary mouse and guinea pig models of JUNV infection. We will evaluate these attenuation strategies in animal models to identify an optimal balance of attenuation and immunogenicity. Our studies will elucidate the molecular basis for rCan attenuation and the immunologic correlates of protection. Strategies targeting the pH-sensing axis in GPC may also be applicable towards the development of an urgently needed live-attenuated Lassa virus vaccine.